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    FAO Nutrition Meetings 
    Report Series No. 48A 
    WHO/FOOD ADD/70.39




    TOXICOLOGICAL EVALUATION OF SOME
    EXTRACTION SOLVENTS AND CERTAIN 
    OTHER SUBSTANCES




    The content of this document is the 
    result of the deliberations of the Joint 
    FAO/WHO Expert Committee on Food Additives 
    which met in Geneva, 24 June  -2 July 19701




    Food and Agriculture Organization of the United Nations
    World Health Organization


                   

    1 Fourteenth report of the Joint FAO/WHO Expert Committee on Food
    Additives, FAO Nutrition Meetings Report Series in press; Wld Hlth
    Org. techn. Rep. Ser., in press.


    CYCLOHEXYLAMINE

    Biological data

    Biochemical aspects

         Following intragastric administration of 5 mg/kg body-weight of
    14 C-CHA to a male rat, 95% was recovered in the first 24 hours, 1.3%
    from cage washings and a trace of 14C-CHA in expired air (but not as 
    14CO2). Activity was evenly divided between urine and faeces.0.22%
    were recovered between 24 and 48 hours. About 85% of the 14C-CHA in
    the urine represented unchanged CHA. In 2 male dogs given oral doses
    of 14C-CHA hydrochloride, more than 90% of the administered dose was
    excreted in the urine within the first 24 hours. Chromatographic
    determinations indicated that an average of 81% of the 14C was CHA
    with an average of 18% representing an unknown metabolite. In a human
    male receiving an oral dose of 14C-CHA about 82% of the dose was
    recovered in the urine within 24 hours, less than 2% being found in
    the faeces. Chromatography indicated that 98% of the 14C in the urine
    was unchanged CHA, 1% was present as an unknown metabolite. 0.6% of
    the 14C remained unidentified (Abbott Laboratories, 1967).

         Orally administered CHA is excreted in the urine of rats to the
    extent of over 99% with less than 1.5% in faeces. 80% of excreted CHA
    is unchanged but 20% appears as unidentified metabolite. CHA is
    rapidly absorbed from the gastrointestinal tract and appears in all
    body tissues (Lethco et al., 1969). CHA-hydrochloride was fed to
    groups of 16 rats at levels of 0, 0.01%, 0.05%, 0.1%, 0.25%, 0.5% and
    1% for 3 months. Only cyclohexylamine was detected in the urine, while
    faeces contained only traces. Blood levels were dose dependent but
    very low, probably because of rapid excretion (Unilever Research
    Laboratories, 1970).

         Following the feeding of 0.17 g/kg of 1-14C-CHA to a rabbit 68%
    of the radioactivity was recovered in the urine in the succeeding 60
    hours. 45% of the administered dose was determined to be unconjugated
    CHA, 0.2% was N-hydroxycyclo-hexylamine (Elliott et al., 1968).

         Incorporation of inorganic S35 into protein polysaccharide was
    used as a test system to show the slight inhibitory effect of CHA on
    membrane processes (Wortman et al., 1968).

    Acute toxicity

                                                                                            

    Animal        Route      LD50 mg/kg      LD100 mg/kg     References
                             body-weight     body-weight
                                                                                        

    Mouse         oral                          500          Lomonova, 1963
    "  (single)   oral          770                          Lee & Dixon, 1969
    "  (groups
       of 10)     oral          520                          Lee & Dixon, 1969
    "             s.c.         1150                          Pliss, 1958
    "             i.p.           50                          Buchel & Guyonneau, 1963
    Rat           oral                          500          Lomonova, 1963
    "             i.p.          200                          Mallette & Von Haam, 1952
    Rabbit        i.p.                          500          Flinn, 1937
    "             dermal                        632          Woodward Labs, 1964
                                                                                        
    
    CHA acts on the spinal motor centres and medulla producing delayed
    convulsions several hours after administration. CHA is partly absorbed
    through the skin but is also a caustic irritant (Carswell & Morrill,
    1937; Lomonova, 1963). A rise in temperature and aggregation of
    animals into groups raises the acute toxicity from 770 mg/kg for
    single mouse at 20 to 445 g/kg for 10 mice at 28C. This effect can
    be prevented by chlorpromazine, and enhanced by SKF-S25A (Lee & Dixon,
    1968; 1969). In man acute inhalation leads to lightheadedness,
    faintness, tachycardia and vomiting (Watrous & Schulz, 1950).

         One-tenth ml of CHA instilled into rabbits' eyes produced
    permanent blindness (Woodward Labs, 1964). After exposure of
    guinea-pigs to a theoretical concentration of 6.7 mg/L of CHA, they
    exhibited salivation, dyspnea, rhinorrhea, ataxia, prostration. 8/10
    guinea-pigs died within 80 minutes. The 2 survivors were observed for
    7 days during which rhonchi were heard and slight rhinorrhea and
    cloudy corneas were noted (Woodward Labs, 1963).

    Special studies

    CHA has been shown to have pressor activity. I.v. injection of doses
    of 0.4 and 3.7 mg/kg produced a sympathomimetic pressor action in
    cats, rats and guinea-pigs (Classen et al, 1968). I.v. injection of 1,
    3 or 10 g/kg CHA into the anaesthetised cat produced a rise in blood
    pressure and tachycardia. These effects were present after vagotomy,
    adrenochetomy, nephrectomy or destruction of the spinal cord. A
    positive inotropic effect was shown in the cat, in isolated perfused
    rat and guinea-pig hearts and in rabbit atria but high doses caused a
    negative inotropic effect. Direct stimulation of skeletal muscle and

    cholinergic stimulation of smooth muscle was demonstrated. The
    sympathomintic effect of CHA were probably mediated through the
    release of endogenous catecholamines and could be absorbed by drugs
    blocking alpha-adrenergic receptors (Rosenblum & Rosenblum, 1968). CHA
    5 mg/kg had no effect on responses to catecholamines, cholinomimetic
    or histamine. Administration of reserpine or phenoxybenzamine
    inhibited the pressor response to CHA. The inhibitory effect of
    reserpine on responses to CHA is reversed by adrenalin infusion.
    Pretreatment with the MAO inhibitor, pargyline had no effect on the
    pressor response to CHA (Hamamura et al., 1968). The effects of CHA on
    the blood pressure are those of an indirectly acting sympathomimetic
    but there is also some direct action on the heart and blood vessels.
    CHA releases H3-labelled noradrenaline from the heart tissue. The cat
    is the oat sensitive animal to the pressor effect of CHA and the
    effects last longer than the rabbit, rat or guinea-pig. The pressor
    response in all 4 species can be blocked by phentolamine (an alpha
    receptor blocking drug). Intragastric or intraduodenal administration
    has also a pressor effect but ten times the i.v. dose is needed.
    Tachyphylaxis has been reported (Unilever Research Laboratories,
    1970). I.v. CHA-sulfate given to dogs at the doses of 3 g/kg produces
    pressor effects by release of noradrenaline from tissue stores
    (Wechsler et al., 1969).

         CHA produces allergic reactions of the delayed hypersensitivity
    type in guinea-pigs (Chung, 1969).

    Short-term studies

         Rat. 12 rats were given 100 mg/kg body-weight/day CHA orally
    for 82 days without obvious adverse effects (Carswell & Morrill,
    1937).

         Groups of 5 males and 5 females were fed diets containing 0, 300
    and 3000 ppm of CHA for 38 weeks. No gross or histopathological
    changes attributable to treatment were noted (Flinn, 1937).

         Guinea-pig. 12 guinea-pigs were given 100 mg/kg body-weight of
    CHA orally for 82 days without obvious adverse effects (Carswell &
    Morrill, 1937).

         Rabbit. 12 rabbits were given 100 mg/kg body-weight of CHA for
    82 days without adverse gross effects (Carswell & Morrill, 1937).

         Dog. Groups of 3 male and 3 female dogs were given by capsule
    0, 0.15, 1.5 and 15 g/kg/day of CHA sulfate. The results based upon 2
    years of feeding do not indicate any CHA effects in terms of
    body-weight, food consumption, mortality, behaviour, haematology and
    chemistry. Gross and microscopic pathology for 1 male and 1 female per
    group sacrificed at 1 year did not indicate any adverse effects. The
    study is still in progress (Industrial Bio-test Lab. Inc., 1969b).

    Long-term studies

         Rat. Groups of 22 males and 28 females were fed 0.5 ml/day of
    5% oily solution of CHA for 12 months and were observed for another 6
    months. No increase in mortality or tumour incidence was noted but
    there were hepatic and renal degenerative changes (Pliss, 1958).

         Four groups of 35 male and 35 female rats were given 0, 0.15
    mg/kg, 1.5 mg/kg, and 15 mg/kg body-weight CHA-sulfate daily for 2
    years. 5 males and 5 females were sacrificed from each group at 52
    weeks. At the end of the study only 75% the animals died from disease
    unrelated to the compound administered. No test-related histological
    abnormalities were seen in the various groups except for one bladder
    tumour in one male rat in the highest dose group, believed to be a
    carcinoma. No other details are available (Oser et al., 1969).

         In a 2 year study in rats, groups of 25 male and 25 female rats
    received CHA sulfate in their diets adjusted so as to provide daily 0,
    0.15, 1-5 and 15 mg/kg. No compound related effects were noted with
    respect to food consumption, mortality, behaviour, haematology,
    chemistry, or organ weights. Males of the 15 mg level showed a reduced
    weight gain during the first 13 months. Microscopic pathology revealed
    urinary bladder changes in 2 rats of the 15 mg/kg group. One female
    sacrificed at 1 year showed epidermoid metaplasia and one male
    sacrificed at 2 years had a transitional cell carcinoma. Bladder
    lesions in these rats are extremely rare (Industrial Bio-test Lab.
    Inc., 1969a).

         In another study, 5 groups of 30 male and 30 female rats were
    given 0, 15, 50, 100 and 150 mg/kg of cyclohexylamine base in their
    diet and will be observed for 2 years. So far at 12 weeks, growth
    depression had been observed in males at 100 and 150 mg/kg levels and
    in females at 5% 100 and 150 mg/kg body-weight levels. These studies
    are being continued (Food and Drug Research Lab., 1970)

    Reproduction teratogenicity studies

         Rat

         In phase 1 of a 3-phase study, groups of 10 male and 20 female
    rats received intragastrically, 0, 1.5 and 15 mg/kg/day of CHA
    sulfate, the males being placed on this regimen beginning at age 30
    days, the females at age 86 days. These animals were mated within
    their groups at 100 days. No effects were noted upon growth, libido,
    or fertility. For females sacrificed at day 14 of pregnancy; control
    and test animals did not differ as to corpora lutea, implantation and
    resorption sites or numbers of viable fetuses. For females allowed to
    go to term, no effects were noted upon gestation time, maternal weight
    or progeny number. Population data, however, showed increased pup
    mortality in the 15 mg/group during the lactation period. The survival
    index at day 4 post-partum for the high dose group and the survival
    indices for both test groups at day 21 were reduced compared to the
    control group. In the teratological phase of this study, groups of 20

    female rats received intragastrically 0, 1.5 and 15 mg/kg/day of CHA
    sulfate on days 6-15. Dams were sacrificed on day 20 and litters
    delivered by caesarean section. Findings revealed no abnormalities
    with respect to growth or mortality of the dams; corpora lutea,
    implantation and resorption sites and fetus viability were normal. No
    abnormalities were seen in the foetuses based upon gross observation,
    free-hand sectioning or alizarin staining. In the third phase, groups
    of 20 pregnant rats received 0, 1.5 and 15 mg/kg/day of CHA sulfate
    beginning on day 15 of pregnancy and continuing through weaning of the
    litters, all of which were cast naturally. While no effects were seen
    with respect to maternal body weight and litter size, the 21-day
    survival and lactation indices for both test groups were lower than
    control indices (Industrial Bio-test Lab. Inc., 1967).

         In two further reproduction and teratology studies essentially
    identical to those already described (Industrial Bio-test Lab. Inc.,
    1967), no effects were seen in one study except for decreased survival
    indices in the high dose level rats during phase 1 (Industrial
    Bio-test Lab. Inc., 1968) and in the second study a questionable
    dose-related decrease in growth during lactation in phase 3 of the
    study (Foster D. Snell, Inc., 1968).

    Chicken

         Fresh fertile eggs were injected directly into yolk or through
    the air cell with 0.2 ppm CHA at 0 and 96 hours of incubation.
    Viability of embryos was reduced and teratogenic effects were noted
    when compared with controls (Verrett, 1969).

    Mutagenicity tests

         CHA was shown to produce chromosomal alterations at certain
    concentrations in microbial cell cultures. The host mediated assay
    using the mouse and S.typhimurium was negative for CHA. In vivo
    cytogenetic analysis of somatic and reproductive cells showed that
    CHA, at 1 mg/kg injected i.p. into the rat daily for 5 days, increased
    chromasom breaks in spermatogonenal cells and 10 mg/kg injected daily
    for 5 days had the same effect on bone marrow cells. The dominant
    lethal test in rats for CHA at 200 mg/kg showed preim-plantation loss
    hence some effect on sperms was possible (US Food and Drug Admin.,
    1969).

         Rats given CHA i.p. showed a dose related incident of chromosomal
    breaks in leucocytes of the bone marrow (Legator et al., I969).

         In groups of 12 rats given 50 mg/kg/day of CHA orally or i.p. for
    5 days no effects on chromosomes of bone marrow cells could be
    demonstrated (Dick & Biava, 1970).
    


    See Also:
       Toxicological Abbreviations
       Cyclohexylamine (ICSC)